The overall goal of this project is to investigate the signaling pathways, particularly ion signaling pathways, that regulate apoptosis. We studied cells that responded differently to apoptotic induction following growth factor removal (i.e. low serum). Early stage preneoplastic, immortal cells (sup+I), show a high susceptibility to induction of apoptosis, whereas late stage preneoplastic cells (sup-II) are relatively resistant to apoptosis following serum reduction to 0.2%. We investigated whether altered Ca2+ homeostasis is causally involved in apoptosis. We showed that a sustained increase in cytosolic free Ca2+ (Cai) does not precede apoptosis. Differences in endoplasmic reticulum (ER) calcium between sup+I cells and sup-II were determined by measuring thapsigargin releasable Ca2+ in the presence of 10% and 0.2% serum. Sup(+) cells in low serum exhibit decreased endoplasmic reticulum calcium levels and subsequent DNA laddering, indicative of apoptosis. The decrease in endoplasmic reticulum calcium in these cells appears to be due, at least in part, to reduced capacitative calcium entry at the plasma membrane. Thus we investigated whether inhibition of capacitative calcium entry per se could reduce endoplasmic reticulum calcium and induce apoptosis of cells. We find that treatment with either SKF96365 (30-100 ?M), an inhibitor of capacitative calcium entry, or cell-impermeant BAPTA (10 mM) is able to induce apoptosis of sup(+) cells in 10% serum, a condition where apoptosis does not normally occur. Because previous work has implicated vesicular trafficking as a mechanism of regulating capacitative calcium entry, we investigated whether disruption of vesicular trafficking could lead to decreased capacitative calcium entry and subsequent apoptosis in sup(+) cells. Coincident with low serum-induced apoptosis, we observed an accumulation of vesicles within the cell, suggesting deregulated vesicle trafficking. Treatment of sup(+) cells with bafilomycin, an inhibitor of the endosomal proton ATPase, produced an accumulation of vesicles within cells without measurably changing cytoplasmic pH. Treatment with 50 nM bafilomycin decreased capacitative entry by ~ 30% and induced apoptosis in sup(+) cells in 10% serum. These data suggest that deregulation of vesicular transport results in reduced capacitative calcium entry which in turn results in apoptosis.